Christine Patte-Mensah

Evidence for a key role of steroids in the modulation of pain

Neurotransmitters such as glutamate, substance P, serotonin and gamma-aminobutyric acid pivotally control pain mechanisms. It is also well known that inflammatory and/or neuropathic pain may depend on the action of diverse cytokines and other molecules including eicosanoids, endorphins, calcitonin-gene related peptide, free radicals and transcription factors. Because steroids control the development, activities and plasticity of the nervous system, these compounds are of particular interest in the modulation of pain. The paper discusses various data supporting the existence of key regulatory effects of steroids in the control of pain. In particular, we analyzed three categories of observations which historically contributed to demonstrate that endogenous and synthetic steroids play a crucial role in the regulation of neurobiological processes involved in pain sensation. The first series of data, which present the chemical characteristics enabling steroids to act on several tissues, also summarize pertinent results supporting the modulation of pain sensation by steroidal compounds. The second category of data evokes psychosocial, fundamental and clinical results suggesting the existence of sex steroid-based differences in pain perception. Finally, we discuss recent evidence showing the endogenous production of neurosteroids and their effects in the spinal cord which crucially controls pain transmission. Taken together, the data reviewed herein suggest that future investigations aiming to develop effective steroid-based strategies against chronic pain must integrate in a complementary manner anti-inflammatory properties of steroids, sex steroid-induced dimorphism in pain perception and regulatory effects exerted by endogenous neurosteroids in pain neural circuits.

Neurogenic pain and steroid synthesis in the spinal cord.

The spinal cord (SC) is a biosynthetic center for neurosteroids, including pregnenolone (PREG), progesterone (PROG), and 3α/5α-tetrahydroprogesterone (3α/5α-THP). In particular, anactive form of cytochrome P450 sidechain cleavage (P450scc) has been localized in sensory networks of the rat SC dorsal horn (DH). P450scc is the key enzyme catalyzing the conversion of cholesterol (CHOL) into PREG, the rate-limiting step in the biosynthesis of all classes of steroids. To determine whether neurosteroidogenesis might be involved in the pivotal role played by the DH in nociception, effects of neurogenic pain provoked by sciatic nerve ligature were investigated on P450scc expression, cellular distribution, and activity in the SC. P450scc mRNA concentration was threefold higher in the DH of neuropathic rats than in controls. The nerve ligature also increased the density of P450scc-positive neuronal perykarya and fibers in the ipsilateral DH. Incubation of spinal tissue homogenates with [3H]CHOL revealed that the amount of newly synthesized [3H]PREG from [3H]CHOL was 80% higher in the DH of neuropathic rats. Radioimmunoassays showed an increase of PREG and 3α/5α-THP concentrations in neuropathic rat DH. The upregulation of PREG and 3α/5α-THP biosynthesis might be involved in endogenous mechanisms triggered by neuropathic rats to cope with the chronic pain state. 3α/5α-THP formation from PREG can also generate PROG, which decreases sensitivity to pain and protects nerve cells against degeneration. Because apoptotic cell death has been demonstrated in the DH during neuropathic pain, activation of neurosteroidogenesis in spinal tissues might also be correlated to the neuroprotective role of steroids in the SC.

Allopregnanolone prevents and suppresses oxaliplatin-evoked painful neuropathy: Multi-parametric assessment and direct evidence

&NA; Oxaliplatin (OXAL) is a platinum‐based drug used for the treatment of colorectal, lung, breast and ovarian cancers. OXAL does not cause renal or hematologic toxicity. However, OXAL induces neuropathic pain which hampers the chemotherapy success. Attempts with neuroprotective agents including anticonvulsivants and antidepressants were made to prevent OXAL‐induced painful neuropathy but the clinical data are controversial and the tested neuroprotectors are able to evoke themselves undesirable effects. Here, we demonstrated that the natural neurosteroid allopregnanolone (3&agr;,5&agr;‐THP), known to be devoid of toxic side‐effects in humans and experimental models, prevented and suppressed OXAL‐induced painful neuropathic symptoms. Indeed, 3&agr;,5&agr;‐THP repaired OXAL‐evoked neurochemical and functional alterations in peripheral nerves and intra‐epidermal nerve fibers (IENF). Behavioral analyses showed that prophylactic or corrective 3&agr;,5&agr;‐THP treatment (4 mg/kg/2 days) respectively prevented or abolished OXAL‐induced cold allodynia, mechanical allodynia and hyperalgesia by reversing to normal decreased thermal and mechanical pain thresholds of OXAL‐treated rats. Electrophysiological investigations revealed that 3&agr;,5&agr;‐THP restored control values of sciatic nerve conduction velocity and action potential peak amplitude drastically reduced by OXAL‐treatment. Furthermore, immunohistochemistry and confocal microscopic quantifications demonstrated that 3&agr;,5&agr;‐THP repaired OXAL‐induced neurochemical/cellular alterations by restoring IENF control density and normal level of neurofilament 200 kDa that was strongly repressed by OXAL in dorsal root ganglion neurons and sciatic nerve axons. OXAL showed no toxicity for the non‐compact myelin protein 2′,3′‐cyclic‐nucleotide‐3′‐phosphodiesterase whose expression level was similarly increased by 3&agr;,5&agr;‐THP in controls and OXAL‐treated rat nerves. Together, these results may be interesting for the development of natural or safe neurosteroid‐based neuroprotective strategy against anticancer drug‐evoked painful neuropathy.

Sciatic nerve injury induces apoptosis of dorsal root ganglion satellite glial cells and selectively modifies neurosteroidogenesis in sensory neurons

Neurosteroids are synthesized either by glial cells, by neurons, or within the context of neuron‐glia cross‐talk. Various studies suggested neurosteroid involvement in the control of neurodegeneration but there is no evidence showing that the natural protection of nerve cells against apoptosis directly depends on their own capacity to produce neuroprotective neurosteroids. Here, we investigated the interactions between neurosteroidogenesis and apoptosis occurring in sensory structures of rats subjected to neuropathic pain generated by sciatic nerve chronic constriction injury (CCI). Using the terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL), we observed no apoptotic cells in the spinal cord up to 30 days after CCI although pain symptoms such as mechano‐allodynia, thermal and mechanical hyperalgesia were evidenced with the Hargreavess behavioral and von Frey filament tests. In contrast, double‐labeling experiments combining TUNEL and immunostaining with antibodies against glutamine synthetase or neuronal nuclei protein revealed apoptosis occurrence in satellite glial cells (SGC) (not in neurons) of CCI rat ipsilateral dorsal root ganglia (DRG) at day 30 after injury. Pulse‐chase experiments coupled with high performance liquid chromatography and flow scintillation detection showed that, among numerous biosynthetic pathways converting [3H]pregnenolone into various [3H]neurosteroids, only [3H]estradiol formation was selectively modified and upregulated in DRG of CCI rats. Consistently, immunohistochemical investigations localized aromatase (estradiol‐synthesizing enzyme) in DRG neurons but not in SGC. Pharmacological inhibition of aromatase caused apoptosis of CCI rat DRG neurons. Altogether, our results suggest that endogenously produced neurosteroids such as estradiol may be pivotal for the protection of DRG sensory neurons against sciatic nerve CCI‐induced apoptosis.

Cellular and functional evidence for a protective action of neurosteroids against vincristine chemotherapy-induced painful neuropathy

Painful neuropathy is a major side-effect limiting cancer chemotherapy. Therefore, novel strategies are required to suppress the neuropathic effects of anticancer drugs without altering their chemotherapeutic effectiveness. By combining biochemical, neuroanatomical/neurochemical, electrophysiological and behavioral methods, we demonstrated that progesterone-derived neurosteroids including 5α-dihydroprogesterone and 3α,5α-tetrahydroprogesterone suppressed neuropathic symptoms evoked in naive rats by vincristine. Neurosteroids counteracted vincristine-induced alterations in peripheral nerves including 2′,3′-cyclic nucleotide 3′-phosphodiesterase, neurofilament-200xa0kDa and intraepidermal nerve fiber repression, nerve conduction velocity, and pain transmission abnormalities (allodynia/hyperalgesia). In skin-tumor rats generated with carcinosarcoma-cells, vincristine, which suppressed the skin tumor and restored normal blood concentration of vascular endothelial growth factor (VEGF), reproduced neuropathic side-effects. Administered alone, neurosteroids did not affect the tumor and VEGF level. Combined with vincristine, neurosteroids preserved vincristine anti-tumor action but counteracted vincristine-induced neural side-effects. Together, these results provide valuable insight into the cellular and functional mechanisms underlying anticancer drug-induced neuropathy and suggest a neurosteroid-based strategy to eradicate painful neuropathy.

Potential role of allopregnanolone for a safe and effective therapy of neuropathic pain.

Because the treatment and management of neuropathic pain are extremely complicated, the characterization of novel analgesics and neuroprotectors with safe toxicological profiles is a crucial need to develop efficient therapies. Several investigations revealed that the natural neurosteroid allopregnanolone (AP) exerts analgesic, neuroprotective, antidepressant and anxiolytic effects. These effects result from AP ability to modulate GABA(A), glycine, L- and T-type calcium channels. It has been shown that AP treatment induced beneficial actions in humans and animal models with no toxic side effects. In particular, a multi-parametric analysis revealed that AP efficiently counteracted chemotherapy-evoked neuropathic pain in rats. It has also been demonstrated that the modulation of AP-producing enzyme, 3α-hydroxysteroid oxido-reductase (3α-HSOR), in the spinal cord regulates thermal and mechanical pain thresholds of peripheral nerve injured neuropathic rats. The painful symptoms were exacerbated by intrathecal injections of provera (pharmacological inhibitor of 3α-HSOR) which decreased AP production in the spinal cord. By contrast, the enhancement of AP concentration in the intrathecal space induced analgesia and suppression of neuropathic symptoms. Moreover, in vivo siRNA-knockdown of 3α-HSOR expression in healthy rat dorsal root ganglia increased thermal and mechanical pain perceptions while AP evoked a potent antinociceptive action. In humans, blood levels of AP were inversely associated with low back and chest pain. Furthermore, oral administration of AP analogs induced antinociception. Altogether, these data indicate that AP, which possesses a high therapeutic potential and a good toxicological profile, may be used to develop effective and safe strategies against chronic neuropathic pain.

Progress in dorsal root ganglion neurosteroidogenic activity: Basic evidence and pathophysiological correlation

Dorsal root ganglia (DRG) which contain glial cells and somas of primary sensory neurons are pivotal for neural transmission between the peripheral and central nervous systems. It is well established that neuropeptides such as substance P and calcitonin gene-related peptide located in DRG neurons control sensory and pain mechanisms. However, contrary to the brain and spinal cord which are extensively investigated, DRG received little attention. Therefore, the current knowledge on DRG may be far to represent their complete neurochemical potential. For instance, until 1997, nothing was known on DRG neurosteroidogenic ability but recently, several investigations have shown that DRG contain various key enzymes synthesizing neuroactive neurosteroids. To provide new advances into DRG neurochemistry, we reviewed and highlighted herein basic and functional evidence showing that neurosteroids are produced in DRG through a neuron-glia crosstalk mechanism. Indeed, key enzymes producing neurosteroids including pregnenolone, progesterone, dihydroprogesterone and estradiol are differentially expressed in DRG cell types. Cytochrome P450side-chain-cleavage is located in DRG neurons and satellite glial cells, 3beta-hydroxysteroid dehydrogenase is expressed in Schwann cells and neurons, 5alpha-reductase is localized in satellite glial and Schwann cells (not in neurons) while aromatase is present in neurons but not in glia. Recent studies also revealed that DRG neurosteroidogenesis is a physiologically relevant process selectively regulated under pathological conditions. Acting through paracrine and autocrine mechanisms, endogenous neurosteroids modulate DRG sensory functions and protect DRG neurons against death. The paper suggests that DRG neurosteroidogenic components may be targeted for the development of therapies against peripheral nerve injury-induced afferent noxious stimulations.

The neuroprotector kynurenic acid increases neuronal cell survival through neprilysin induction

Kynurenic acid (KYNA), one of the main product of the kynurenine pathway originating from tryptophan, is considered to be neuroprotective. Dysregulation of KYNA activity is thought to be involved in neurodegenerative diseases, the physiopathology of which evokes excitotoxicity, oxidative stress and/or protein aggregation. The neuroprotective effect of KYNA is generally attributed to its antagonistic action on NMDA receptors. However, this single target action appears insufficient to support KYNA beneficial effects against complex neurodegenerative processes including neuroinflammation, β-amyloid peptide (Aβ) toxicity and apoptosis. Novel insights are therefore required to elucidate KYNA neuroprotective mechanisms. Here, we combined cellular, biochemical, molecular and pharmacological approaches to demonstrate that low micromolar concentrations of KYNA strongly induce neprilysin (NEP) gene expression, protein level and enzymatic activity increase in human neuroblastoma SH-SY5Y cells. Furthermore, our studies revealed that KYNA exerts a protective effect on SH-SY5Y cells by increasing their viability through a mechanism independent from NMDA receptors. Interestingly, KYNA also induced NEP activity and neuroprotection in mouse cortical neuron cultures the viability of which was more promoted than SH-SY5Y cell survival under KYNA treatment. KYNA-evoked neuroprotection disappeared in the presence of thiorphan, an inhibitor of NEP activity. NEP is a well characterized metallopeptidase whose deregulation leads to cerebral Aβ accumulation and neuronal death in Alzheimers disease. Therefore, our results suggest that a part of the neuroprotective role of KYNA may depend on its ability to induce the expression and/or activity of the amyloid-degrading enzyme NEP in nerve cells.

Selective regulation of 3α-hydroxysteroid oxido-reductase expression in dorsal root ganglion neurons: A possible mechanism to cope with peripheral nerve injury-induced chronic pain

&NA; The enzyme 3&agr;‐hydroxysteroid oxido‐reductase (3&agr;‐HSOR) catalyzes the synthesis and bioavailability of 3&agr;,5&agr;‐neurosteroids as allopregnanolone (3&agr;,5&agr;‐THP) which activates GABAA receptors and blocks T‐type calcium channels involved in pain mechanisms. Here, we used a multidisciplinary approach to demonstrate that 3&agr;‐HSOR is a cellular target the modulation of which in dorsal root ganglia (DRG) may contribute to suppress pain resulting from peripheral nerve injury. Immunohistochemistry and confocal microscope analyses showed 3&agr;‐HSOR‐immunostaining in naive rat DRG sensory neurons and glial cells. Pulse‐chase, high performance liquid chromatography and Flo/One characterization of neurosteroids demonstrated 3&agr;,5&agr;‐THP production in DRG. Behavioral methods allowed identification of pain symptoms (thermal and mechanical hyperalgesia and/or allodynia) in rats subjected to sciatic nerve chronic constriction injury (CCI). Reverse transcription and real‐time polymerase chain reaction revealed that 3&agr;‐HSOR mRNA concentration in CCI‐rat ipsilateral DRG, 5‐fold higher than in contralateral DRG, was also 4‐ to 6‐fold elevated than that in sham‐operated or naive rat DRG. Consistently, Western blotting confirmed increased 3&agr;‐HSOR protein levels in CCI‐rat ipsilateral DRG and double immunolabeling showed that 3&agr;‐HSOR overexpression occurred in DRG neurons but not in glia. Functional plasticity of 3&agr;‐HSOR leading to increased 3&agr;,5&agr;‐THP production was evidenced in CCI‐rat DRG. Interestingly, behavioral and molecular time‐course investigations revealed that 3&agr;‐HSOR gene upregulation was correlated to pain symptom development. Most importantly, in vivo knockdown of 3&agr;‐HSOR expression in healthy rat DRG using 6‐carboxyfluorescein‐3&agr;‐HSOR‐siRNA exacerbated thermal and mechanical pain perceptions. This paper is the first to show that siRNA‐induced knockdown of a key neurosteroid‐synthesizing enzyme directly affects an important function as nociception. Hopefully, these results may be useful for the development of novel analgesics.

Local modulation of steroid action: rapid control of enzymatic activity

Estrogens can induce rapid, short-lived physiological and behavioral responses, in addition to their slow, but long-term, effects at the transcriptional level. To be functionally relevant, these effects should be associated with rapid modulations of estrogens concentrations. 17β-estradiol is synthesized by the enzyme aromatase, using testosterone as a substrate, but can also be degraded into catechol-estrogens via hydroxylation by the same enzyme, leading to an increase or decrease in estrogens concentration, respectively. The first evidence that aromatase activity (AA) can be rapidly modulated came from experiments performed in Japanese quail hypothalamus homogenates. This rapid modulation is triggered by calcium-dependent phosphorylations and was confirmed in other tissues and species. The mechanisms controlling the phosphorylation status, the targeted amino acid residues and the reversibility seem to vary depending of the tissues and is discussed in this review. We currently do not know whether the phosphorylation of the same amino acid affects both aromatase and/or hydroxylase activities or whether these residues are different. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues.